c. Description The Biomedical Research Infrastructure (BRI) is an underground two floor, 120,000 square foot, 27,000 cage animal facility. This state-of-the-art automated facility is in the courtyard of the Biological Laboratories, which places it adjacent to the Northwest Building. The BRI houses a Genome Modification Facility (GMF) in an 800 square feet procedural room in the BRI with additional animal holding space for 1,000 cages in adjacent rooms. This room is equipped with 4 vibration free microinjection stations, micro-needle fabrication equipment, 4 banks of tissue culture incubators for embryo/ES cell culture, 3 tissue culture hoods, 3 laminar flow hoods for pathogen free minor surgeries, 12 stereomicroscopes of which 2 will be equipped with GFP optics and teaching video, automatic liquid nitrogen storage facilities, and computers. The GMF as a whole is governed by faculty committee of four individuals: Andrew McMahon (Molecular and Cellular Biology, Committee Chair), Joshua Sanes (CBS), and Kevin Eggan (HSCI). The committee oversees effective running of the GMF. The GMF staff consists of the following. The director, Manfred Baetscher, PhD, has broad experimental experience of genetic modification of mice and many years of managerial experience. He participates directly in production of genetically modified strains, remains current on emerging genetic approaches, and advises investigators on appropriate genetic strategies for their experiments. Transgenic research assistants: Two assistants, both with proven experience in either or both pronuclear injection of DMA and blastocyst injection and small animal surgery. Tissue culture technicians: A full time technician, soon to be hired will perform all aspects of ES cell culture, ES cell electroporation and clonal selection and DMA preparation forgenotyping. Animal husbandry: Two animal care technicians conduct all support functions for embryo generation and biopsy preparation. Administrative assistant: This individual is responsible for all ordering, billing and coordination between investigators and GMF. Finally, it is worth noting that the BRI will also contain behavioral testing suites equipped for phenotypic analysis of genetically modified mice, including models of neurological and psychiatric diseases. These will be supported from other sources, so are not described here, but it is our intention to integrate them with the GMF. The GMF provides the following services: Production of transgenic mice by pronuclear injection of DMA constructs in plasmids, BACs, or YACs ES cell targeting and injection to generate knock-out and knock-in mice Cryopreservation and storage of embryos and sperm for valuable strains not in current use Mouse strain resuscitation by in vitro fertilization and intracytoplasmic sperm injection (ICSI) Re-derivation of mouse strains by embryo transfer to render them pathogen-free The development of lentiviral vector based transgenes Consultation on genetic model creation Plans to offer gene targeting in embryonic stem cells and validation of DNA vectors and feeder lines We also hope the GMF will provide new functionality that we will add if this grant is funded, circumventing a bottleneck that many users of genetically modified mice encounter. There are a dozen or so strains that are highly useful for a broad variety of neuroscience applications. Most are publicly available but the waiting time to obtain them collaborators or stock centers (e.g., at Jackson Laboratories) is often 6 months or more, and the expense is considerable. Yet, few laboratories can afford to maintain stocks permanently of lines that may need only once every year or two. Lines in this category include: (a) Mice in which specific neuronal populations are marked with GFP or one of its spectral variants (e.g., motoneurons in YFP-16, layer 5 pyramids in YFP-H, very small numbers of forebrain neurons in GFP-M, or inhibitory interneurons in GAD65- GFP). (b) Mice in which expression of a reporter is conditional on expression of ere recombinase (e.g., Z/EG, Z/AP and thy1-stop-YFP). (c) Mice in which ere or flp recombinase is expressed in the neural tube generally (e.g., Nestin-Cre) or in specific populations of neurons (e.g., CaM kinase-Cre) or only following activation by tamoxifen (e.g., CAGS-CreER). So, we propose to maintain small colonies (approximately 4 cages of 4 males each) of each of about a dozen lines. The lines will be chosen by the Steering Committee. In most cases, the mice are already available in at least one user laboratory. Then, when any Harvard neuroscientist requires one of the lines, we will be able to provide two males within a week, and then replenish our own supply by breeding. We believe this repository will greatly decrease the barrier to use of valuable strains, thereby making possible risky or pilot experiments that would otherwise be prohibitively expensive or unduly slow.